Antistreptococcal group a antibodies: Production after in vitro activation and hybridization of mouse spleen cells

Abstract

Streptococcal group A polysaccharide-specific antibodies were raised by the method of somatic cell hybridization. Spleen cells of experimentally unprimed BALB/c and C57BL/6 mice were activated in vitro by the streptococcal vaccine and fused with the Sp2/0-Ag14 line at times 0, 35, 70, and 105 h thereafter. Hybridomas were obtained at all times independent of the addition of thymocyte-conditioned medium. Occurrence of specific hybridomas for the T cell-dependent A-CHO, however, required activation for greater than 35 h. Low responder C57BL/6 splenocytes fused at considerably higher fusion efficiency to yield specific hybridomas than high responder spleen cells 105 h after activation by antigen. The isotypes of A-CHO-specific antibodies comprised predominantly μ and k polypeptides; however, γ3, α, and λ polypeptide chains were also identified. All specific antibodies were agglutinating the group A streptococcal cells; this agglutination was fully inhibited by the addition of 1 % N-acetyl-D-glucosamine, the immune determinant sugar of the A-CHO. Three hybridomas obtained by fusion of BALB/c splenocytes 105 h after activation were cloned and grown as tumours in the peritoneal cavity of BALB/c mice. The monoclonal antibodies in the ascites did not precipitate the A-CHO but continued to agglutinate group A streptococcal cells in a hapten inhibitable fashion with different specificity profiles. Antibody from clone 21S36.1 was coprecipitable upon addition of A-CHO with a γG3 monoclonal hybridoma-derived antibody in a ratio of sol1 7 while the other two monoclonal γM antibodies and the S117 myeloma protein were not. This result suggests that antibody 21S36.1 recognizes one chain terminal determinant of the A-CHO.